Method of detecting salmonella typhimurium

ABSTRACT

Provided herein are methods and compositions for detecting  Salmonella Typhimurium  in a sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Application 62/351,130 filedon Jun. 16, 2016 which is hereby incorporated by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically herewith and is hereby incorporated byreference in its entirety. The ASCII copy, created on Jun. 7, 2017, isnamed 1050901_SEQ_ST25.txt, and is 5,026 bytes in size.

BACKGROUND

Salmonella is a leading cause of foodborne illnesses worldwide, withpoultry and pork products being a primary source of infection to humans.Detecting Salmonella can be challenging because low levels of thebacteria may not be recovered using traditional culturing techniques.The genus Salmonella, member of the Enterobacteriaceae family, comprisestwo species Salmonella enterica and Salmonella bongori. Salmonellaenterica is further divided into six subspecies, of which S. entericasubsp. enterica is the most clinically significant, causing 99% ofSalmonella infections. The subspecies are further sub-divided into morethan 2,500 serovars defined by somatic and flagellar antigens.Salmonella enterica subsp. enterica serovar Typhimurium and Salmonellaenterica subsp. enterica serovar Enteritidis are the most frequentlyreported serovars associated with human cases of Salmonella infectionfrom foodborne outbreaks. In the EU, a regulation in force since 2003governs the mandatory detection of Salmonella. In 2011, this regulationwas supplemented with the mandatory testing for S. Enteritidis and S.Typhimurium. According to Commission Regulation (EU) No. 1086/2011, allfresh poultry must be examined for S. Enteritidis and S. Typhimuriumcontamination. In the United States, the Food and Drug Administration(FDA) has published the Final Rule “Prevention of Salmonella Enteritidisin Shell Eggs During Production, Storage, and Transportation” (74 FR33030), which will introduce methods requiring egg producers to test forS. Enteritidis. For non-egg producers, the FDA also published theguidance document for testing of human foods for salmonella: “Guidancefor Industry: Testing for Salmonella Species in Human Foods andDirect-Human-Contact Animal Foods”.

Conventional microbiological methods for the detection andidentification of Salmonella serovars are very time consuming. Thecurrent accepted method for isolation of Salmonella from food andenvironmental primary production samples takes up to 5 days according tothe ISO 6579. The most widely-used method used to characterizeSalmonella into its subspecies is the Kauffman-White serotyping system,based on the variability of the O, H and Vi antigens.

SUMMARY

Described herein are methods and compositions for detecting SalmonellaTyphimurium.

In an embodiment, a method of selectively detecting the presence ofSalmonella Typhimurium in a sample comprises (a) providing a reactionmixture comprising a suitable primer pair for amplification of residues749 to 2136 (1388 bp), or a portion thereof, of Salmonella TyphimuriumACCESSION CP007235 (SEQ ID NO:1); (b) performing PCR amplification ofthe nucleic acids of the sample using the reaction mixture of step (a);and (c) selectively detecting the presence of Salmonella Typhimurium bydetecting the amplified nucleic acids. In some embodiments, the step (b)is performed in partitions. In some embodiments, the detecting thepresence of Salmonella Typhimurium comprises sequencing the amplifiednucleic acids.

In some embodiments, the reaction mixture comprises a primer pair foramplification of a sequence 95%, 97% or 99% homologous to SEQ ID NO:1 ora portion thereof. In certain embodiments, the reaction mixturecomprises a primer pair for amplification of residues 749 to 1697 (947bp), or portions thereof, of Salmonella Typhimurium ACCESSION CP007235(SEQ ID NO:2). In some embodiments, the reaction mixture comprises aprimer pair for amplification of a sequence 95%, 97% or 99% homologousto SEQ ID NO:2 or a portion thereof. In certain embodiments, thereaction mixture comprises a primer pair for amplification of residues755 to 1063 (309 bp), or portions thereof, of Salmonella TyphimuriumACCESSION CP007235 (SEQ ID NO:3). In some embodiments, the reactionmixture comprises a primer pair for amplification of a sequence 95%, 97%or 99% homologous to SEQ ID NO:3 or a portion thereof.

In an embodiment, the primer pair for amplification of the nucleic acidregion of SEQ ID NO:3 comprises the polynucleotide sequences set forthin SEQ ID NO:4 and SEQ ID NO:5. In an embodiment, the reaction mixturefurther comprises a probe for the nucleic acid region to be detected. Insome embodiments, the probe comprises a detectable label. In someembodiment, the probe comprises the polynucleotide sequences set forthin SEQ ID NO:6 and SEQ ID NO:7. In certain embodiments, the probecomprises the polynucleotide sequences set forth in SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, or SEQ ID NO:9.

In an embodiment, an isolated polynucleotide comprises a polynucleotidesequence having at least 95% sequence identity based on the BLASTNmethod of alignment to the polynucleotide sequence set forth in SEQ IDNO:1. In some embodiments, the isolated polynucleotide sequencecomprises a polynucleotide sequence set forth in SEQ ID NO:1. In anembodiment, an isolated polynucleotide comprises a polynucleotidesequence having at least 95% sequence identity based on the BLASTNmethod of alignment to the polynucleotide sequence set forth in SEQ IDNO:2. In some embodiments, the isolated polynucleotide sequencecomprises a polynucleotide sequence set forth in SEQ ID NO:2. In anembodiment, an isolated polynucleotide comprises a polynucleotidesequence having at least 95% sequence identity based on the BLASTNmethod of alignment to the polynucleotide sequence set forth in SEQ IDNO:3. In some embodiments, the isolated polynucleotide sequencecomprises a polynucleotide sequence set forth in SEQ ID NO:3.

In an embodiment, a kit for the detection of Salmonella Typhimurium in asample comprises a primer pair comprising SEQ ID NO:4 and SEQ ID NO:5.In some embodiments, the kit further comprises a probe comprising SEQ IDNO:6 and SEQ ID NO:7. In some embodiments, the kit further comprises aprobe comprising comprising SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, orSEQ ID NO:9. In certain embodiments, the kit further comprises at leastone component selected from a lysis reagent, a DNA polymerase, at leastone dNTP, a buffer, a negative control, a positive control, andinstructions for performing a method to detect the presence ofSalmonella Typhimurium in a nucleic acid sample.

DETAILED DESCRIPTION

Provided herein are methods of selectively detecting the presence ofSalmonella Typhimurium in a sample. Also provided are compositions foruse in the detection of Salmonella Typhimurium in a sample by nucleicacid amplification, e.g., by real-time PCR.

The disclosed detection method finds utility in the detection of S.Typhimurium in any type of sample, for example in samples for foodtesting, environmental testing, or human/animal diagnostic testing.Exemplary food samples include, but are not limited to, meats products,poultry (e.g., chicken, turkey), eggs, fish (e.g, cod), cookie dough,produce (e.g, lettuce, tomatoes), dairy (e.g, cheese, milk), milk powder(e.g., infant formula), chocolate (e.g., milk), cocoa, nacho cheeseseasoning, pasta, pet food, peanut butter, soy flour, spices, andready-to-eat food. Environmental samples include, but are not limitedto, plastic, sealed concrete, and stainless steel. Other types ofsamples include, but are not limited to, water, stool, blood, urine, andtissue. Another type of sample includes weeds. The methods may beperformed at the farm or processing facility prior to initial packaging,after packaging (e.g., prior to or after export from one country toanother), or at the point of sale.

I. DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4^(th) ed. 2007); Green et al., MOLECULAR CLONING, ALABORATORY MANUAL (FOURTH EDITION), Cold Spring Harbor Laboratory Press(Cold Spring Harbor, N.Y. 2012).

The term “a” or “an” is intended to mean “one or more.” The term“comprise” and variations thereof such as “comprises” and “comprising,”when preceding the recitation of a step or an element, are intended tomean that the addition of further steps or elements is optional and notexcluded. Any methods, devices and materials similar or equivalent tothose described herein can be used in the practice of this invention.The following definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

The term “nucleic acid” refers to polymers of deoxyribonucleotides orribonucleotides in either single- or double-stranded form, andcomplements thereof. The term “polynucleotide” refers to a linearsequence of nucleotides. Nucleotides can be ribonucleotides,deoxyribonucleotides, or modified versions thereof. Examples ofpolynucleotides contemplated herein include single and double strandedDNA, single and double stranded RNA (including siRNA), and hybridmolecules having mixtures of single and double stranded DNA and RNA.

“Polymerase chain reaction” is abbreviated PCR.

The term “isolated” refers to materials, such as nucleic acid moleculesand/or proteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides can bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans can be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment” are used interchangeably herein.These terms encompass nucleotide sequences and the like. Apolynucleotide can be a polymer of RNA or DNA that is single- ordouble-stranded, that optionally contains synthetic, non-natural, oraltered nucleotide bases. A polynucleotide in the form of a polymer ofDNA can be comprised of one or more strands of cDNA, genomic DNA,synthetic DNA, or mixtures thereof.

The term “amplification product” refers to nucleic acid fragmentsproduced during a primer-directed amplification reaction. Typicalmethods of primer-directed amplification include polymerase chainreaction (PCR), ligase chain reaction (LCR), or strand displacementamplification (SDA). If PCR methodology is selected, the replicationcomposition can comprise the components for nucleic acid replication,for example: nucleotide triphosphates, two (or more) primers withappropriate sequences, thermostable polymerase, buffers, solutes, andproteins.

The term “primer” refers to a synthetic oligonucleotide that is capableof acting as a point of initiation of nucleic acid synthesis orreplication along a complementary strand when placed under conditions inwhich synthesis of a complementary strand is catalyzed by a polymerase.A primer can further contain a detectable label, for example a 5′ endlabel.

The term “probe” refers to a synthetic oligonucleotide that iscomplementary (though not necessarily fully complementary) to apolynucleotide of interest and forms a duplexed structure byhybridization with at least one strand of the polynucleotide ofinterest. A probe can further contain a detectable label.

As used herein, the terms “label”, “detectable label”, and such refer toa molecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes,metal ions, metal sols, semiconductor nanocrystals, and ligands (e.g.,biotin, avidin, streptavidin, or haptens). A detectable label can alsoinclude a combination of a reporter and a quencher.

The term “reporter” refers to a substance or a portion thereof which iscapable of exhibiting a detectable signal, which signal can besuppressed by a quencher. The detectable signal of the reporter is,e.g., fluorescence in the detectable range; thus, a reporter can also bea label.

The term “quencher” refers to a substance which is capable ofsuppressing, reducing, inhibiting, etc., the detectable signal producedby the reporter.

As used herein, the term “quenching” refers to a process whereby, when areporter and a quencher are in close proximity, and the reporter isexcited by an energy source, a substantial portion of the energy of theexcited state non-radiatively transfers to the quencher where it eitherdissipates nonradiatively or is emitted at a different emissionwavelength than that of the reporter (e.g., by fluorescence resonanceenergy transfer or FRET).

The reporter can be selected from fluorescent organic dyes modified witha suitable linking group for attachment to the oligonucleotide, such asto the terminal 3′ carbon or terminal 5′ carbon. The quencher can alsobe selected from organic dyes, which may or may not be fluorescent,depending on the embodiment of the invention. Generally, whether thequencher is fluorescent or simply releases the transferred energy fromthe reporter by non-radiative decay, the absorption band of the quenchershould at least substantially overlap the fluorescent emission band ofthe reporter to optimize the quenching.

Non-fluorescent quenchers or dark quenchers typically function byabsorbing energy from excited reporters, but do not release the energyradiatively.

Selection of appropriate reporter-quencher pairs for particular probescan be undertaken in accordance with known techniques. Fluorescent anddark quenchers and their relevant optical properties from whichexemplary reporter-quencher pairs can be selected are listed anddescribed, for example, in R. W. Sabnis, HANDBOOK OF FLUORESCENT DYESAND PROBES, John Wiley and Sons, New Jersey, 2015, the content of whichis incorporated herein by reference.

Reporter-quencher pairs can be selected from xanthene dyes includingfluoresceins and rhodamine dyes. Many suitable forms of these compoundsare available commercially with substituents on the phenyl groups, whichcan be used as the site for bonding or as the bonding functionality forattachment to an oligonucleotide. Another group of fluorescent compoundsfor use as reporters are the naphthylamines, having an amino group inthe alpha or beta position. Included among such naphthylamino compoundsare 1-dimethylaminonaphthyl-5 sulfonate, 1-anilino-8-naphthalenesulfonate and 2-p-touidinyl-6-naphthalene sulfonate. Other dyes include3-phenyl-7-isocyanatocoumarin; acridines such as9-isothiocyanatoacridine; N-(p-(2-benzoxazolyl)phenyl)maleimide;benzoxadiazoles; stilbenes; pyrenes and the like.

Suitable examples of quenchers can be selected from6-carboxy-tetramethyl-rhodamine, 4-(4-dimethylaminophenylazo) benzoicacid (DABCYL), tetramethylrhodamine (TAMRA), BHQ-OTM, BHQ-1 TM, BHQ-2TM,and BHQ-3TM, each of which are available from Biosearch Technologies,Inc. of Novato, Calif., Qy7TM QSY-9TM, QSY-21 TM and QSY-35TM, each ofwhich are available from Molecular Probes, Inc, Iowa Black™ FQ availablefrom Integrated DNA Technologies.

Suitable examples of reporters can be selected from dyes such as SYBRgreen, 5-carboxyfluorescein (5-FAMTm available from Applied Biosystemsof Foster City, Calif.), 6-carboxyfluorescein (6-FAM),tetrachloro-6-carboxyfluorescein (TET),2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein,hexachloro-6-carboxyfluorescein (HEX),6-carboxy-2′,4,7,7′-tetrachlorofluorescein (6-TETTm available fromApplied Biosystems), carboxy-X-rhodamine (ROX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (6-JOETM availablefrom Applied Biosystems), VICTM dye products available from MolecularProbes, Inc., NEDTM dye products available from Applied Biosystems, CalFluor dye products (such as, e.g., Cal Fluor Gold 540, Orange 560, Red590, Red 610, Red 635) available from Biosearch Technologies, Quasar dyeproducts (such as, e.g., Quasar 570, 670, 705) available from BiosearchTechnologies, and the like.

The term “percent identity,” in the context of two or more nucleicacids, refers to two or more sequences or subsequences that are the sameor have a specified percentage of nucleotides that are the same (i.e.,about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parameters, or bymanual alignment and visual inspection. See e.g., the NCBI web site atncbi.nlm.nih.gov/BLAST. Such sequences are then said to be“substantially identical.” Percent identity is typically determined overoptimally aligned sequences, so that the definition applies to sequencesthat have deletions and/or additions, as well as those that havesubstitutions. The algorithms commonly used in the art account for gapsand the like. Typically, identity exists over a region comprising asequence that is at least about 25 nucleotides in length, or over aregion that is 50-100 nucleotides in length, or over the entire lengthof the reference sequence.

The terms “selectively” or “selective” with respect to nucleic acidsrefers to the discrimination between the target nucleic acid sequence(e.g., target sequence of Salmonella Typhimurium) over the non-targetnucleic acid sequences (e.g., non-target sequence SalmonellaTyphimurium). An assay is selective for a sequence if little or nohybridization of the primer or probe occurs with non-target sequence.

The terms “partitioning” or “partitioned” refer to separating an aqueoussolution having one or more of a sample and reactant into a plurality ofportions, or “partitions.” Partitions can be solid or fluid. In someembodiments, a partition is a solid partition, e.g., a microchannel. Insome embodiments, a partition is a fluid partition, e.g., a droplet. Insome embodiments, a fluid partition (e.g., a droplet) is a mixture ofimmiscible fluids (e.g., water and oil). In some embodiments, a fluidpartition (e.g., a droplet) is an aqueous droplet that is surrounded byan immiscible carrier fluid (e.g., oil).

II. NUCLEIC ACIDS Genome Detection Regions

A detection method is provided herein that is based on theidentification of residues 749 to 2136 (1388 bp) of SalmonellaTyphimurium ACCESSION CP007235 (see SEQ ID NO: 1 in Table 1).

TABLE 1 Sequences SEQ ID NO DESCRIPTION SEQUENCE 1 1388 bp fragmentATGTAGCTTAAGATATCTATAGTGATATCAGTGTAATACTTATTGGTTAG from the 3315 bpATCGGTATGATCTTGAATATTTTTATATCGATAGTTTGGATTACATAGTA gene ofGAGTTATTTCACTTTGCAATACAGCTTTAATTATAGTTTTGTCAAGTTGT SalmonellaAATTTATCTATAAAAATATTATTTATAGTATTTTCTATTAGGAGAAGTGT TyphimuriumTTCGACTAACTTGATATTTGTATTGATTTTTTGTTTGTAGATATTCCGTA ACCESSIONGCAATTGAGTTGAATTGTGTTCAAGCAATGGTGAACAAACATAATCCCAT CP007235GATTGCTCTTGAGAGTCCCAGTCATTTTTAGCTATTTCAATAGCATTGGTGACTAATTCGATAATTTCATCTTCAATTTCTGGATATGGTACTGAGGCTAATTCACCACTAGTAAAGCTAAGTGTGGGGGCTAGTATTGATAAATAATGGTTTACAACCGGAGTGCACATTAATCCCGCAGCGTAAAGCAACTCATTTTTGTTATTTGAAAAGCCGCAACGGCCTGTATCATCAAAAACAAACCCTTTTGGACGATATCTCACGCAAAAATTACCTTGGCTTATTTTTGACCATGTTATGCCTTCTCTAAAGTAATACTCATCATTTCTTACGGCAGAGCGAGTTTTGCCATTCTCAAATTTAAAATTTCGTATTTCGTAACCATTATTTTCCCAATTTACAACTATTTCGTTATTACCATACCACTTTCGATATTCACCTCCACTACTACAAGGAAACCATTTGATATTATGAATGTCGATTTTTGTATTTGATTCTTTATTTGTGATAAGGGTTTTTTTTATTGAAACCTCGTACCAATATCTTTGAAATTTAATATTGTCACCGGTGGACATGCCTGCTTTTAATGCTATTTTTTCTCCAAGTTTTTTATGGTGGCGAAAAGATAATAGACTCGGTAAGTCTATCCAATATGCTATTGGCATTCCTGGTATGTTTTTAAAATCATGCTGTGTAAATTTATCAAATATATTTTTCCTTAGAAGTAGATCGCTTTTCTTTACTTCTTCCCTACCATCTATAAGTCTAAAAAATACAGGTTGGTAACGTTCGGAGTGTTGGTTTTTAATCACCCAGGCAGTTGTCTGTACAACCTCTCCAGAAATTTGCCCAAAAGCCCGAGCTCCCAAATGTGCCATCGTAATAAATGTTTTATTGTCCAATAACCAGTTACGTAGTGCTTCATAACTTGACAAAAACATCCATGATTGCATATTGACTTGAGCATTAAACCCATTTTCTTTAAGCAAAGAAAATGCATTCTGCATAAACATTGCAAACAAATCAGCTTTACTATCCGGGAAGTTATTTTTGGCAAACTCTTTCAGCTCACTATTCATTCCCTTGCC 2 947 bp fragmentATGTAGCTTAAGATATCTATAGTGATATCAGTGTAATACTTATTGGTTAG of SalmonellaATCGGTATGATCTTGAATATTTTTATATCGATAGTTTGGATTACATAGTA TyphimuriumGAGTTATTTCACTTTGCAATACAGCTTTAATTATAGTTTTGTCAAGTTGT ACCESSIONAATTTATCTATAAAAATATTATTTATAGTATTTTCTATTAGGAGAAGTGT CP007235TTCGACTAACTTGATATTTGTATTGATTTTTTGTTTGTAGATATTCCGTAGCAATTGAGTTGAATTGTGTTCAAGCAATGGTGAACAAACATAATCCCATGATTGCTCTTGAGAGTCCCAGTCATTTTTAGCTATTTCAATAGCATTGGTGACTAATTCGATAATTTCATCTTCAATTTCTGGATATGGTACTGAGGCTAATTCACCACTAGTAAAGCTAAGTGTGGGGGCTAGTATTGATAAATAATGGTTTACAACCGGAGTGCACATTAATCCCGCAGCGTAAAGCAACTCATTTTTGTTATTTGAAAAGCCGCAACGGCCTGTATCATCAAAAACAAACCCTTTTGGACGATATCTCACGCAAAAATTACCTTGGCTTATTTTTGACCATGTTATGCCTTCTCTAAAGTAATACTCATCATTTCTTACGGCAGAGCGAGTTTTGCCATTCTCAAATTTAAAATTTCGTATTTCGTAACCATTATTTTCCCAATTTACAACTATTTCGTTATTACCATACCACTTTCGATATTCACCTCCACTACTACAAGGAAACCATTTGATATTATGAATGTCGATTTTTGTATTTGATTCTTTATTTGTGATAAGGGTTTTTTTTATTGAAACCTCGTACCAATATCTTTGAAATTTAATATTGTCACCGGTGGACATGCCTGCTTTTAATGCTATTTTTTCTCCAAGTTTTTTATGGTGGCGAAAAGATAATAGACTCGGTAAGTCTAT 3 123 bp fragmentTAGGAGAAGTGTTTCGACTAACTTGATATTTGTATTGATTTTTTGTTTGT of SalmonellaAGATATTCCGTAGCAATTGAGTTGAATTGTGTTCAAGCAATGGTGAACAA TyphimuriumACATAATCCCATGATTGCTCTTG ACCESSION CP007235 4 Forward PrimerTAGGAGAAGTGTTTCGACTAAC for 123 bp target sequence spanning residues937 to 958 5 Reverse Primer CAAGAGCAATCATGGGATTATG for 123 bp targetsequence spanning residues 1038 to 1059 6 Probe for 123 bpTTTACAATTGAGTTGAATTGTGTTCAAGC target sequence spanning residues1000 to 1024 (5′FAM-3′BkFQ) 7 Probe for 123 bpAAAAGAACACAATTCAACTCAATTGCTACG target sequence spanning residues995 to 1020 (5′FAM-3′BkFQ) 8 Probe for 123 bp CAATTGAGTTGAATTGTGTTCAAGCtarget sequence spanning residues 1000 to 1024 (5′FAM-3′BkFQ) 9Probe for 123 bp GAACACAATTCAACTCAATTGCTACG target sequencespanning residues 995 to 1020

Based on a publicly available software (e.g., BLAST), SEQ ID NO:1 isconserved (e.g., 100% sequence identity) in 672 Salmonella Typhimuriumstrains listed by Genbank accession number in Table 2.

TABLE 2 Strains of Salmonella Typhimurium in which SEQ ID NO: 1 is 100%Conserved LVHC01000004.1 LAPF01000001.1 CTPI01000002.1 CTHF01000003.1LVHA01000006.1 LAPD01000063.1 CTPH01000002.1 CTHE01000002.1LVGZ01000004.1 LAPC01000018.1 CTPG01000002.1 CTHD01000002.1LVGY01000004.1 LAOX01000013.1 CTPF01000002.1 CTHC01000003.1LVGX01000005.1 JZAH01000029.1 CTPE01000003.1 CTHB01000001.1LVGW01000004.1 JZAB01000025.1 CTPD01000001.1 CTHA01000002.1LVGU01000047.1 JYZR01000005.1 CTPC01000003.1 CTGZ01000002.1LVGT01000051.1 JYZC01000010.1 CTPB01000003.1 CTGY01000002.1LVGS01000046.1 JYZB01000030.1 CTPA01000004.1 CTGX01000005.1LVGR01000047.1 JYYZ01000004.1 CTOZ01000002.1 CTGW01000025.1LVGQ01000013.1 JYYU01000025.1 CTOY01000002.1 CTGV01000002.1LVGP01000004.1 JYYT01000016.1 CTOX01000003.1 CTGU01000006.1LVGN01000006.1 JYYQ01000066.1 CTOW01000001.1 CTGT01000002.1LVGM01000008.1 JYYL01000011.1 CTOV01000003.1 CTGS01000002.1LVGL01000007.1 JYYK01000018.1 CTOU01000002.1 CTGR01000004.1LVGK01000003.1 JYYH01000035.1 CTOT01000003.1 CTGQ01000002.1LVGJ01000009.1 JYYC01000006.1 CTOS01000002.1 CTGP01000004.1LVGI01000006.1 JYYB01000003.1 CTOR01000003.1 CTGO01000001.1LVGH01000005.1 JYYA01000024.1 CTOQ01000004.1 CTGN01000001.1LVGG01000006.1 JYXZ01000019.1 CTOP01000003.1 CTGM01000002.1LVGF01000006.1 JYXX01000004.1 CTOO01000002.1 CTGL01000004.2LVFW01000011.1 JYXW01000003.1 CTON01000002.1 CTGK01000002.1LVFV01000011.1 JYXT01000019.1 CTOM01000004.1 CTGJ01000035.1LVFT01000012.1 JYXK01000009.1 CTOL01000003.1 CTGI01000004.1LVFS01000014.1 JYXH01000002.1 CTOK01000002.1 CTGH01000004.1LVFR01000005.1 JYXC01000008.1 CTOJ01000001.1 CTGG01000002.1LVFQ01000008.1 JYWW01000034.1 CTOI01000002.1 CTGF01000002.1LVFP01000006.1 JYWU01000014.1 CTOH01000003.1 CTGE01000002.1LVFO01000002.1 JYWT01000009.1 CTOG01000003.1 CTGD01000002.1LVFN01000004.1 JYWA01000003.1 CTOF01000002.1 CTGC01000002.1LVFM01000005.1 JYVW01000007.1 CTOE01000002.1 CTGB01000004.1LVFL01000037.1 JYVV01000004.1 CTOD01000002.1 CTGA01000002.1LVFK01000003.1 JYVU01000035.1 CTOC01000003.1 CTFZ01000004.1LVFJ01000013.1 JYVL01000001.1 CTOB01000002.1 CTFY01000002.1LVFI01000003.1 JYVJ01000004.1 CTOA01000004.1 CTFX01000001.1LVFH01000010.1 JYVG01000055.1 CTNZ01000003.1 CTFW01000003.1LUJG01000006.1 JYVE01000037.1 CTNY01000003.1 CTFV01000004.1LUJF01000005.1 JYUZ01000004.1 CTNX01000002.1 CTCJ01000002.1LUJE01000003.1 JYUY01000010.1 CTNW01000004.1 CTCI01000003.1LUJC01000020.1 JYUP01000020.1 CTNV01000003.1 CTGH01000003.1LUIZ01000005.1 JYTX01000005.1 CTNU01000002.1 CTCG01000002.1LUIY01000005.1 JYTW01000027.1 CTNT01000002.1 CTCF01000002.1LUIW01000007.1 JYTU01000018.1 CTNS01000003.2 CTCE01000003.1LUIU01000061.1 JYTT01000012.1 CTNR01000001.1 CTCD01000002.1UIT01000019.1 JYTQ01000001.1 CTNQ01000001.1 CTCC01000044.1 UIS01000029.1JYTP01000017.1 CTNP01000002.1 CTCB01000003.1 LUIR01000027.1JYTL01000012.1 CTNO01000002.1 CTCA01000003.1 LUIQ01000009.1JYTI01000010.1 CTNN01000003.1 CTBZ01000002.1 LUIP01000019.1JYTD01000002.1 CTNM01000002.1 CTBY01000002.1 LUIO001000023.1JYTB01000004.1 CTNL01000002.1 CTBX01000003.1 LUIN01000035.1JYSY01000001.1 CTNK01000004.1 CTBW01000004.1 LUIM01000025.1JYSX01000031.1 CTNJ01000001.1 CTBV01000003.1 LUIL01000040.1JYSO01000094.1 CTNI01000002.1 CTBU01000002.1 LUIK01000041.1JYSN01000051.1 CTNH01000002.1 CTBT01000001.1 LUIJ01000024.1JYSM01000007.1 CTNG01000036.1 CTBS01000003.1 LUII01000036.1JYSL01000029.1 CTNF01000003.1 CTBR01000004.1 LUIH01000014.1JYSG01000016.1 CTNE01000002.1 CTBQ01000003.1 LUIG01000040.1JYSB01000024.1 CTND01000002.1 CTBP01000005.1 LUIF01000036.1JYSA01000003.1 CTNC01000001.1 CTBO01000002.1 LUIE01000017.1JYRX01000002.1 CTNB01000046.1 CTBN01000003.1 LUID01000024.1JYRW01000014.1 CTNA01000004.1 CTBM01000003.1 LUIC01000116.1JYRT01000085.1 CTMZ01000002.1 CTBK01000003.1 LUIB01000040.1JYRQ01000034.1 CTMY01000004.1 CTBJ01000002.1 LUIA01000027.1JYRM01000039.1 CTMX01000002.1 CTBI01000002.1 LUHZ01000025.1JYRD01000017.1 CTMW01000004.1 CTBH01000003.1 LUHY01000038.1JYRC01000016.1 CTMV01000003.1 CTBF01000004.1 LUHX01000025.1JYQU01000020.1 CTMU01000003.1 CTBE01000004.1 LUHV01000033.1JYQM01000015.1 CTMT01000004.1 CTBC01000002.1 LUHU01000040.1JYQC01000008.1 CTMS01000002.1 CTBB01000002.1 LUHT01000028.1JYQA01000030.1 CTMR01000004.1 CTBA01000002.1 LUHS01000024.1JYPT01000005.1 CTMQ01000003.1 CTAZ01000003.1 LUHR01000036.1JYPP01000050.1 CTMP01000002.1 CTAY01000001.1 LONA01000003.1JYPO01000064.1 CTMO01000003.1 CTAX01000001.1 LKJI01000024.1JYPM01000008.1 CTMN01000003.1 CTAW01000001.1 LKJE01000032.1JUIT01000021.1 CTMM01000003.1 CTAV01000002.1 LKJD01000006.1JRZW01000002.1 CTML01000002.1 CTAU01000001.1 LJJK01000144.1JRZV01000001.1 CTMK01000002.1 CTAT01000003.1 LIOJ01000001.1JRZU01000002.1 CTMJ01000003.1 CTAS01000002.1 LIOG01000016.1JRZT01000030.1 CTMI01000002.1 CTAR01000002.1 LIOF01000021.1JRZS01000015.1 CTMH01000003.1 CTAQ01000002.1 LIOB01000013.1JRZR01000010.1 CTMG01000002.1 CTAP01000002.1 LINM01000004.1JRZQ01000006.1 CTMF01000002.1 CTAO01000002.1 LINL01000011.1JRZO01000062.1 CTME01000001.1 CTAN01000001.1 LINK01000002.1JRZN01000054.1 CTMD01000003.1 CTAM01000004.1 LINJ01000020.1JRZM01000003.1 CTMC01000004.1 CTAL01000003.1 LINF01000009.1JRZL01000059.1 CTMB01000002.1 CTAK01000004.1 LINE01000010.1JRZK01000010.1 CTMA01000002.1 CTAJ01000002.1 LINB01000005.1SJRZJ01000012.1 CTLZ01000002.1 CTAI01000002.1 LIMZ01000030.1SJRZI01000017.1 CTLY01000002.1 CTAH01000003.1 LIMV01000008.1JRZH01000001.1 CTLX01000004.1 CTAG01000004.1 LIMU01000022.1JRYU01000004.1 CTLW01000002.1 CTAF01000002.1 LIMT01000027.1JRYT01000009.1 CTLV01000004.1 CTAE01000002.1 LIMN01000012.1JRGW01000022.1 CTLU01000001.1 CTAD01000002.1 LIMM01000010.1JRGV01000045.1 CTLT01000004.1 CTAC01000004.1 LIMH01000001.1JRGU01000009.1 CTLS01000004.1 CTAB01000002.1 LIDY01000003.1JRGT01000034.1 CTLR01000003.1 CQIE01000002.1 LHOM01000023.1JRGS01000001.1 CTLQ01000001.1 CQHW01000002.1 LHOJ01000006.1JRGR01000006.1 CTLP01000003.1 CQHV01000002.1 LHOH01000004.1JHAI01000005.1 CTLO01000001.1 CQHU01000002.1 LHOG01000003.1JHAH01000020.1 CTLN01000003.1 CQHS01000002.1 LHOF01000033.1JHAG01000020.1 CTLM01000004.1 CQHR01000002.1 LHNW01000001.1JHAF01000018.1 CTLL01000004.1 CQHQ01000002.1 LHNS01000017.1JHAE01000013.1 CTLK01000002.1 CQHN01000002.1 LHNM01000038.1FKJD01000009.1 CTLJ01000002.1 CQHJ01000001.1 LHNL01000010.1FKJC01000004.1 CTLI01000002.1 CQHI01000002.1 LHNI01000015.1FKJB01000003.1 CTLH01000004.1 CQHH01000002.1 LHNH01000007.1CYID01000003.1 CTLG01000004.1 CQHG01000001.1 LHNG01000002.1CYIC01000003.1 CTLF01000002.1 CQHF01000001.1 LHMT01000026.1CYIB01000003.1 CTLE01000002.1 CQHE01000002.1 LHMS01000025.1CYIA01000002.1 CTLD01000004.1 CQHD01000002.1 LHMA01000007.1CYHZ01000003.1 CTLC01000006.1 CQHC01000002.1 LHLX01000009.1CYHY01000003.1 CTLB01000002.1 CQHB01000002.1 LHLU01000004.1CYHX01000003.1 CTLA01000002.1 CQHA01000001.1 LHLP01000001.1CYHW01000002.1 CTKZ01000002.1 CQGZ01000001.1 LHLO01000016.1CYHV01000002.1 CTKI01000002.1 CQGY01000002.1 LHLK01000019.1CYHU01000003.1 CTKG01000001.1 CQGX01000002.1 LHLF01000020.1CYHT01000003.1 CTKD01000002.1 CQGW01000002.1 LHLA01000001.1CVMK01000002.1 CTKC01000002.1 CQGV01000002.1 LHKL01000042.1CVMH01000002.1 CTKB01000004.1 CQGU01000002.1 LHKK01000034.1CVKN01000003.1 CTKA01000003.1 CQGT01000001.1 LHKD01000036.1CVKM01000005.1 CTJZ01000002.1 CQGS01000002.1 LHKC01000003.1CVKK01000004.1 CTJY01000002.1 CQGR01000002.1 LHKA01000038.1CVKJ01000005.1 CTJX01000003.1 CQGQ01000002.1 LHJU01000003.1CVKH01000004.1 CTJW01000002.1 CQGP01000002.1 LHJJ01000028.1CVKC01000002.1 CTJV01000003.1 CQGO01000002.1 LHJF01000017.1CVKA01000004.1 CTJU01000005.1 CQGN01000002.1 LHIX01000004.1CVJW01000001.1 CTJT01000002.1 CQGM01000002.1 LHIW01000017.1CTQZ01000003.1 CTJS01000002.1 CQGL01000002.1 LHIR01000013.1CTQY01000002.1 CTJR01000003.1 CQGK01000001.1 LHIQ01000047.1CTQX01000004.1 CTJQ01000002.1 CQGJ01000002.1 LHIK01000014.1CTQU01000002.1 CTJP01000002.1 CQGI01000001.1 LHIE01000015.1CTQT01000003.1 CTJO01000004.2 CQGH01000002.1 LHID01000040.1CTQS01000002.1 CTJN01000001.1 CQGG01000002.1 LHHK01000011.1CTQR01000002.1 CTJM01000004.1 CIJW01000002.1 LHHB01000001.1CTQQ01000001.1 CTJK01000003.1 CIJD01000002.1 LHGZ01000002.1CTQP01000002.1 CTJJ01000002.1 CHJY01000001.1 LHGY01000001.1CTQO01000002.1 CTIL01000002.1 CGGH01000001.1 LHGL01000047.1CTQN01000002.1 CTIK01000003.1 CGDA01000002.1 LHGJ01000005.1CTQM01000002.1 CTIJ01000003.1 CGCS01000001.1 LHGI01000006.1CTQL01000098.1 CTII01000002.1 CGCQ01000002.1 LHGH01000003.1CTQK01000004.1 CTIH01000002.1 CGCH01000002.1 LHFW01000031.1CTQJ01000002.1 CTIG01000002.1 CFOA01000002.1 LHFV01000044.1CTQI01000001.1 CTIF01000004.1 CFNX01000002.1 LHFU01000031.1CTQH01000001.1 CTIE01000003.1 CFLD01000002.1 LHFT01000032.1CTQG01000002.1 CTID01000002.1 BAKU01000002.1 LHFS01000022.1CTQF01000003.1 CTIC01000002.1 AYVJ01000114.1 LHFP01000043.1CTQE01000004.1 CTIB01000003.1 AYUQ01000108.1 LHFF01000013.1CTQD01000002.1 CTIA01000004.1 AUXR01000008.1 LHEY01000021.1CTQC01000001.1 CTHZ01000002.1 AUXE01000003.1 EHEX01000008.1CTQB01000003.1 CTHY01000001.1 AUVE01000003.1 LHEW01000017.1CTQA01000002.1 CTHX01000001.1 AUVD01000043.1 LHEV01000005.1CTPZ01000003.1 CTHW01000001.1 AUQU01000045.1 LHER01000004.1CTPY01000003.1 CTHV01000002.1 AUQT01000034.1 LHEQ01000025.1CTPX01000004.1 CTHU01000002.1 AUQO01000062.1 LHEP01000049.1CTPW01000002.1 CTHT01000004.1 AUQN01000039.1 LHEA01000005.1CTPV01000002.1 CTHS01000002.1 AUQM01000038.1 LFGM01000013.1CTPU01000005.1 CTHR01000002.1 AOXO01000096.1 LFDY01000028.1CTPT01000001.1 CTHQ01000002.1 AOXD01000065.1 LFDW01000010.1CTPS01000002.1 CTHP01000002.1 AOXC01000083.1 LFCC01000059.1CTPR01000003.1 CTHO01000004.1 AJTU01000007.1 LDPA01000008.1CTPQ01000002.1 CTHN01000002.1 AHVA01000018.1 LAPQ01000066.1CTPP01000002.1 CTHM01000002.1 AHUZ01000018.1 LAPP01000078.1CTPO01000002.1 CTHL01000002.1 AHUV01000085.1 LAPO01000058.1CTPN01000002.1 CTHK01000002.1 AHUT01000031.1 LAPN01000091.1CTPM01000005.1 CTHJ01000001.1 AHUS01000047.1 LAPM01000076.1CTPL01000004.1 CTHI01000039.1 AERV01000023.1 LAPH01000042.1CTPK01000003.1 CTHH01000002.1 ABAO01000006.1 LAPG01000091.1CTPJ01000002.1 CTHG01000002.1 LUIV01000062.1

Also based on a publicly available software (e.g., BLAST), SEQ ID NO:1is partially conserved (e.g., at least 99% sequence identity) in 48Salmonella Typhimurium strains listed by Genbank accession number inTable 3.

TABLE 3 Strains of Salmonella Typhimurium in which SEQ ID NO: 1 isPartially Conserved LVGO01000006.1 LUJA01000002.1 LDYH01000006.1AMEA02000013.1 LVGE01000003.1 LUIX01000003.1 JYPX01000009.1AMDZ02000027.1 LVGD01000004.1 LFGR01000011.1 JTED01000004.1AMDY01000216.1 LVGC01000003.1 LFGQ01000013.1 AUQV01000023.1AMDX02000108.1 LVGB01000001.1 LFGP01000017.1 ATWR01000084.1JRZX01000002.1 LVGA01000003.1 LFGN01000015.1 AMEH02000182.1JRZP01000003.1 LVFZ01000005.1 LFDX01000009.1 AMEG02000055.1AUQL01000040.1 LVFY01000001.1 LFCI01000010.1 AMEF02000044.1LFGO01000009.1 LVFX01000003.1 LFCH01000028.1 AMEE02000031.1LFDZ01000012.1 LVFU01000005.1 LFCG01000008.1 AMED02000059.1LECA01000009.1 LUJD01000002.1 LECD01000013.1 AMEC02000055.1LUHW01000058.1 LUJB01000005.1 LECC01000010.1 AMEB02000105.1LECB01000012.1

Based on a publicly available software (e.g., BLAST), SEQ ID NO:1 is notfound in the following non-Typhimurium Salmonella strains:

Salmonella_enterica_serovar_Ohio_CFSAN001079Salmonella_enterica_serovar_Newport_WA14881Salmonella_enterica_serovar_Schwarzengrund_SL480Salmonella_enterica_serovar_Gallinarum_28791Salmonella_enterica_serovar_Thompson_2010K1863Salmonella_enterica_serovar_Kentucky_CDC191Salmonella_enterica_serovar_Typhi_BL196Salmonella_enterica_serovar_Enteritidis_P125109Salmonella_enterica_serovar_Agona_72A52Salmonella_enterica_serovar_Saintpaul_JO2008Salmonella_enterica_serovar_Heidelberg_SL476Salmonella_enterica_serovar_Virchow_ATCC51955Salmonella_enterica_serovar_Paratyphi_B_SARA62Salmonella_enterica_serovar_MontevideoMDA09249507Salmonella_enterica_serovar_Muenchen_2009K0951Salmonella_enterica_serovar_B areilly_CFSAN000197Salmonella_enterica_serovar_Virchow_SL491Salmonella_enterica_serovar_Pullorum_19945Salmonella_enterica_serovar_Dublin_SL1438Salmonella_enterica_serovar_Stanley_060538

In another embodiment, a detection method is based on the identificationof residues 749-1697 of Salmonella Typhimurium ACCESSION CP007235REGION: 658819 . . . 662133 (see SEQ ID NO:2 in Table 1). In anotherembodiment, a detection method is based on the identification ofresidues 755-1063 of Salmonella Typhimurium ACCESSION CP007235 REGION:658819 . . . 662133 (see SEQ ID NO:3 in Table 1). In some embodiments,the detection method incorporates unlabeled primers and labeled probesfor the detection of Salmonella Typhimurium.

Oligonucleotides

Oligonucleotides of the instant invention are set forth in SEQ ID NOs:4-9.

Disclosed oligonucleotides can be used as primers for PCR amplificationand as hybridization probes. Primers and probes are shown in Table 1.

The nucleic acid probes can contain a detectable label. In someembodiments, the probe comprises a reporter-quencher combination asemployed in a double-stranded probe, a TAQMAN™ probe, a molecular beaconprobe, a SCORPION™ probe, a dual hybridization probe, or an ECLIPSE™probe. In some embodiments, a double-stranded probe comprises twocompletely or partially complementary strands. In some embodiments, onestrand of the double-stranded probe comprises a reporter on the 5′ endand the other strand comprises a quencher on the 3′ end such that whenthe two strands hybridize, the reporter and quencher face each other andthe quencher quenches the fluorescence emitted by the reporter. DuringPCR, the strands separate, allowing the reporter to fluoresce and to bedetected. In some embodiments, each strand of the double-stranded probeincludes a reporter at one end (e.g., the 5′ end) and a quencher at theother end (e.g., the 3′ end). When the two strands hybridize with eachother, the reporter from the first strand is in close proximity with thequencher of the second strand such that fluorescence quenching occurs.During PCR, the strands separate, allowing the reporter to fluoresce andto be detected. In an embodiment, the probe is a double-stranded probeas described in U.S. Pat. No. 9,194,007, which is incorporated byreference in its entirety herein. In an embodiment, a reporter-quencherpair used in a double-stranded probe is 6-FAM and Iowa Black® FQ.

III. METHODS

The oligonucleotides can be used in a method for selectively detectingthe presence of Salmonella Typhimurium in a sample. In an embodiment,the method begins by providing a reaction mixture comprising a suitableprimer pair for amplification of residues 749-1697, or a portionthereof, of SEQ ID NO:2. In some embodiments, the reaction mixturecomprises a primer pair for amplification of a sequence 95%, 97%, or 99%homologous to SEQ ID NO:2. In some embodiments, the reaction mixturecomprises a primer pair for amplification of a sequence of SEQ ID NO:2or a portion thereof.

In certain embodiments, the reaction mixture comprises a primer pair foramplification of residues 755-1063, or portions thereof, of SEQ ID NO:3.In some embodiments, the reaction mixture comprises a primer pair foramplification of a sequence 95%, 97%, or 99% homologous to SEQ ID NO:3.In some embodiments, the reaction mixture comprises a primer pair foramplification of a sequence of SEQ ID NO:3 or a portion thereof. In someembodiments, the primer pair for amplification of the nucleic acidregion of SEQ ID NO:3 comprises SEQ ID NO:4 and SEQ ID NO:5.

In some embodiments, the method further comprises a probe for thenucleic acid region to be detected. In certain embodiments, the probecomprises a detectable label. In some embodiments, the probe is asingle-stranded probe comprising SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,or SEQ ID NO:9. In some embodiments, each probe is labeled with areporter on one end (e.g., the 5′ end) and a quencher on the other end(e.g., the 3′ end). In some embodiments, the probe is a double-strandedprobe comprising SEQ ID NO:6 and SEQ ID NO:7 (e.g., SEQ ID NO:6 canhybridize to SEQ ID NO:7) with each strand having a reporter on one end(e.g., the 5′ end) and a quencher on the other end (e.g., the 3′ end).

The next step of the method comprises performing PCR amplification(e.g., real-time PCR) of the nucleic acids of the sample using thereaction mixture. In some embodiments, PCR amplification is performed inpartitions (e.g, droplets). Methods and compositions for partitioning asolution are described, for example, in published patent applications WO2012/135259, WO 2014/117088, WO 2010/036352, and U.S. Pat. No.9,156,010, the entire content of each of which is incorporated byreference herein.

In the last step of the method, the presence of Salmonella Typhimuriumis selectively detected by detecting the amplified nucleic acids. Insome embodiments, the detecting step comprises sequencing the amplifiednucleic acids.

IV. KITS

In another aspect, kits for detecting Salmonella Typhimurium in a sampleaccording to the methods described herein are provided. In someembodiments, a kit comprises a primer pair as described herein. In someembodiments, the kit further comprises probes as described herein. Insome embodiments, the kit further comprises assay components including,but not limited to, a lysis reagent, a DNA polymerase, dNTPs, a buffer,a negative control, and a positive control. In some embodiments, the kitfurther comprises instructions for carrying out the methods describedherein.

V. EXAMPLES Example 1—Comparison of Salmonella Typhimurium Assay of theInstant Invention to Salmonella Spp. Assay

In this example, the Salmonella Typhimurium assay of the instantinvention was compared to a commercially available Salmonella spp.Assay. In the experiment, eleven Salmonella serovars that are mostrelevant for the food industry were tested with the SalmonellaTyphimurium assay of this disclosure and with the iQ-Check Salmonellaspp. II Assay (Bio-Rad). The eleven Salmonella strains were streaked ona TCS Petri dish and allowed to grow for 24 hr at 37° C. Individualcolonies were then picked, diluted in 500 μL sterile water and 5 μL weretested with each assay using the Bio-Rad CFX96 Touch™ Real-Time PCRDetection System. For the Salmonella Typhimurium assay, thedouble-stranded probe comprised SEQ ID NOs 6 and 7. Each strand of thedouble-stranded probe was labeled with 6-FAM on the 5′ end and IowaBlack™ FQ on the 3′end. The double-stranded probe was synthesized byIntegrated DNA Technologies using phosphoramidite chemistry. Results areshown in Table 4.

TABLE 4 Comparison of Assays Bio-Rad iQ-Check Salmonella spp. II assaySalmonella Typhimurium assay Target Internal Target Internal Serovars Cqcontrol Cq Result Cq control Cq Result Negative Ctrl N/A 32.84 NegativeN/A 32.49 Negative Positive Ctrl 31.72 32.24 Positive 31.95 31.78Positive Typhimurium 19.69 34.60 Positive 19.36 33.43 PositiveMonophasic Typhimurium 18.35 N/A Positive 18.08 34.2 PositiveEnteritidis 20.34 32.72 Positive N/A 32.15 Negative Infantis 20.86 32.40Positive N/A 31.78 Negative Virchow 19.71 33.28 Positive N/A 32 NegativeHadar 21,20 32.29 Positive N/A 31.89 Negative Paratyphi B Java 20.2433.27 Positive N/A 31.91 Negative Livingstone 20.84 33.21 Positive N/A32.38 Negative Kentucky 18.67 34.26 Positive N/A 32.17 Negative Dublin21,24 32.68 Positive N/A 31.99 Negative Newport 20.23 32.67 Positive N/A31.98 Negative

The results shown in Table 4 illustrate that only typhimurium isdetected by the Salmonella Typhimurium assay. The results also show thatthe sensitivities of both assays are identical and that the SalmonellaTyphimurium assay can be used as a primary screening assay or as aconfirmatory, serotyping assay.

Example 2—Assay Selectivity

This example illustrates assay selectivity of the instant invention.One-hundred and nine Salmonella enterica subsp. enterica serovars andSalmonella enterica subspecies (in italics in Table 4) were tested withthe Salmonella Typhimurium assay. The same method and probes as inExample 1 were used in this experiment. The organisms tested are shownin Table 5.

TABLE 5 Selectivity Abaetetuba Aberdeen Adelaïde Agama Albany Anatumarizonae* Bambylor Bareilly Berta Betioky Blegdam Blockley bongoriBraenderup Brandenburg Bredeney Budapest California Cerro Carrau CanogaCrossness Cubana Choleraesuis diarizonae* Dalhem Derby Dublin EmekDuisberg Enteritidis Fischerkietz Ferruch Give Gaminara GallinarumGlostrup Grumpensis Grabow Goldgoast Havana Hadar Guinea Havannahoutenae* Illinois Heidelberg Indiana indica* Inverness JohannesburgInfantis Kentucky Kirkee Kottbus Kedougou Lomita Livingstone ManicaLondon Miami Minnesota Maregrosso Mbandaka Muenchen Montevideo MoscowNapoli Nienstedten Naestved Newport Nottingham Oranienburg Ouakam OkatieOhio Phoenix Panama Paratyphi B** Paratyphi B java Postdam Poona PuttinQuentin Rostock Salamae Rubislaw Senftenberg Saint Paul SchwarzengrundSingapore Sheffield Sundsvall Springs Strasbourg Taksony TallahasseeTournai Tenessee Thompson Treforest Tranoroa Utrecht Virchow ZuerichYoruba Wayne Worthington

Of the organisms listed in Table 5, all but Paratyphi B were notdetected with the assay. The results illustrate that the SalmonellaTyphimurium assay is highly selective for Salmonella Typhimurium.

Example 3—Assay Exclusivity

This example illustrates assay exclusivity of the instant invention.Thirty-nine non-Salmonella bacteria were tested with the SalmonellaTyphimurium assay. The same method and probe as in Example 1 was used inthis experiment. The bacteria tested are tabulated in Table 6. None ofthe bacteria listed in Table 6 were detected by the SalmonellaTyphimurium assay, illustrating assay exclusivity.

TABLE 6 Exclusivity Acinetobacter baumanii Aeromonas hydrophilaAeromonas hydrophila/caviae Bacillus licheniformis Bacillus cereusCampylobacter jejuni Campylobacter coli Campylobacter lari Campylobacterupsaliensis Citrobacter freundii Cronobacter sakazakii Enterobactercloacae Enterobacter pyrinus Enterobacter sakazakii Enterobacteraerogenes Enterobacter asburiae Enterobacter amnigenus Enterobactercowanii Enterococcus faecium Escherichia coli Escherichia hermaniiHafnia alvei Klebsiella oxytoca Klebsiella pneumoniae Listeriamonocytogenes Micrococcus luteus Pantoea agglomerans Proteus mirabilisPseudomonas fluorescens Pseudomonas aeruginosa Raoultella terrigenaSerratia marcescens Shigella flexneri Shigella sonnei Staphylococcusaureus Staphylococcus internmedius Staphylococcus xylosus Staphylococcusepidermidis Yersinia enterocoloitica

Example 4—Assay Specificity

This example illustrates assay specificity of the instant invention.Seventy-nine Salmonella Typhimurium serovars were tested with theSalmonella Typhimurium assay. The same method and probe as in Example 1was used in this experiment. The bacteria tested are tabulated in Table7. All of the bacteria listed in Table 7 were detected by the SalmonellaTyphimurium assay, illustrating assay specificity.

TABLE 7 Specificity Antigenic Primary Strain number Strain numberSerovar formula Comment source Origin (Bio-Rad Library) (Other Library)Typhimurium 1,4,[5],12:i:1,2 Anses Brine 002 no Anses: 38.09 Typhimurium1,4,[5],12:i:1,2 Anses Beef meat 003 no Anses: 442.09 Typhimurium1,4,[5],12:i:1,2 Anses Pork (crépine 004 no Anses: 447.09 de porc)Typhimurium 1,4,[5],12:i:1,2 Anses Lamb with 005 no Anses: 591.09 sauceTyphimurium 1,4,[5],12:i:1,2 Anses Stuffed quail 006 no Anses: 695.09Typhimurium 1,4,[5],12:i:1,2 Anses Culture from 007 no Anses: 839.09lamb feces Typhimurium 1,4,[5],12:i:1,2 Anses Environment 008 no Anses:838.09 (Duck) Typhimurium 1,4,[5],12:i:1,2 Anses Culture from 009 noAnses: 553.11 horse feces Typhimurium 1,4,[5],12:i:1,2 Anses Whitepepper 010 no Anses: 564.11 Typhimurium 1,4,[5],12:i:1,2 Anses Hokifilet with 011 no Anses: 708.11 cream Typhimurium 1,4,[5],12:i:1,2 AnsesPigeon viscera 012 no Anses: 781.11 Typhimurium 1,4,[5],12:i:1,2 AnsesCompost 013 no Anses: 792.11 Typhimurium 1,4,[5],12:i:1,2 AnsesEnvironmental 014 no Anses: 835.11 (Chicken) Typhimurium1,4,[5],12:i:1,2 Anses Streaky ham 015 no Anses: 840.11 Typhimurium1,4,[5],12:i:1,2 Anses Whole quail 016 no Anses: 845.11 Typhimurium1,4,[5],12:i:1,2 Anses Spareribs 017 no Anses: 880.11 Typhimurium1,4,[5],12:i:1,2 Anses Culture from 018 no Anses: 886.11 swine fecesTyphimurium 1,4,[5],12:i:1,2 Anses Raw milk 019 no Anses: 907.11 (cow)Typhimurium 1,4,[5],12:i:1,2 Anses Sausage 020 no Anses: 976.11Typhimurium 1,4,[5],12:i:1,2 Anses Tomato filling 021 no Anses: 977.11Typhimurium 1,4,[5],12:i:1,2 Anses Stuffed 022 no Anses: 979.11 potatoesTyphimurium 1,4,[5],12:i:1,2 Anses Fish meal 023 no Anses: 985.11Typhimurium 1,4,[5],12:i:1,2 Anses Pet food 043 no Anses: 1175.11Typhimurium 1,4,[5],12:i:1,2 Anses Foie gras 030 no Anses: 119.11(Liver) Typhimurium 1,4,[5],12:i:1,2 ADRIA Milk powder 081 ADRIA no4Development Typhimurium 1,4,[5],12:i:1,2 ADRIA Pasteurized 082 ADRIAno13 Development liquid egg Typhimurium 1,4,[5],12:i:1,2 ADRIAPasteurized 083 ADRIA no206 Development liquid egg Typhimurium1,4,[5],12:i:1,2 ADRIA Egg yolk 084 ADRIA no472 Development Typhimurium1,4,[5],12:i:1,2 ADRIA Pasteurized 085 ADRIA no776 Development liquidegg Typhimurium 1,4,[5],12:i:1,2 ADRIA Ready-to-eat 086 CIP 58.58Development Typhimurium 1,4,[5],12:i:1,2 ADRIA Foie (Liver) 087 ADRIAno19 Development Typhimurium 1,4,[5],12:i:1,2 ADRIA Raw ground 088 ADRIAno22 Development meat Typhimurium 1,4,[5],12:i:1,2 ADRIA Ready-to-eat089 ADRIA no167 Development Typhimurium 1,4,[5],12:i:1,2 ADRIAChipolatas 090 ADRIA no193 Development sausages Typhimurium1,4,[5],12:i:1,2 ADRIA Chipolatas 091 ADRIA no830 Development sausagesTyphimurium 1,4,[5],12:i:1,2 ADRIA Merguez 092 ADRIA no911 Developmentsausages Typhimurium 1,4,[5],12:i:1,2 ADRIA Chipolatas 093 ADRIA no987Development sausages Typhimurium 1,4,[5],12:i:1,2 ADRIA Meat (pâté) 094ADRIA no4874 Development Typhimurium 1,4,[5],12:i:1,2 ADRIA Frozen meat095 A00C003 Development Typhimurium 1,4,[5],12:i:1,2 ADRIA Frozen meat096 A00C004 Development Typhimurium 1,4,[5],12:i:1,2 ADRIA Frozen beef097 A00C059 Development trim Typhimurium 1,4,[5],12:i:1,2 ADRIA groundbeef 098 A00C060 Development Typhimurium 1,4,[5],12:i:1,2 ADRIA Pork 106Ad1070 Development Typhimurium 1,4,[5],12:i:1,2 ADRIA Pork 107 ST325Development Typhimurium 1,4,[5],12:i:1,2 ADRIA Pork 108 ST1 DevelopmentTyphimurium 1,4,[5],12:i:1,2 ADRIA Pork 109 ST394 DevelopmentTyphimurium 1,4,[5],12:i:1,2 ADRIA Pork 110 ST719 DevelopmentTyphimurium 1,4,[5],12:i:1,2 ADRIA Pork 111 ST11 Development Typhimurium1,4,[5],12:i:1,2 ADRIA Liquid egg 113 JES411 Development Typhimurium1,4,[5],12:i:1,2 ADRIA Beef trim 116 Ad913 Development Typhimurium1,4,[5],12:i:1,2 ADRIA Pork 118 Ad1249 Development Typhimurium1,4,[5],12:i:1,2 ADRIA pork (crépine) 119 Ad1338 Development Typhimurium1,4,[5],12:i:1,2 ADRIA ground meat 120 Ad1410 Development Typhimurium1,4,[5],12:i:1,2 ADRIA Liquid egg 121 Ad1484 Development Typhimurium1,4,[5],12:i:1,2 ADRIA Drinking 122 Ad1546 Development water from troughTyphimurium 1,4,[5],12:i:1,2 ADRIA salmon with 123 Ad1603 Developmentvegetables Typhimurium 1,4,[5],12:—:— non motile ADRIA Tiramisu 124Ad1333 variant Development Typhimurium 1,4,[5],12:—:1,2 monophasic ADRIAHen 125 Ad1335 variant Development Typhimurium 1,4,[5],12:i:— monophasicADRIA Pork specialty 126 Ad1334 variant Development Typhimurium1,4,[5],12:i:1,2 Anses Environmental 160 2002LSAL00347 (Quail)Typhimurium 1,4,[5],12:i:1,2 Anses Environmental 161 2016LSAL02607(goose) Typhimurium 1,4,[5],12:i:— monophasic Anses Environmental 1622009LSAL04410 variant (Pork) Typhimurium 1,4,[5],12:—:1,2 monophasicAnses Environmental 163 2010LSAL01759 variant (Gallus gallus- hen)Typhimurium 1,4,[5],12:i:— monophasic Anses Environmental 1642011LSAL04681 variant Typhimurium 1,4,[5],12:i:— monophasic AnsesEnvironmental 165 2012LSAL04635 variant (Turkey) Typhimurium1,4,[5],12:i:1,2 Anses Environmental 166 2013LSAL00987 (Gallus gallus)Typhimurium 1,4,[5],12:i:— monophasic Anses Environmental 1672014LSAL00857 variant (Bovine) Typhimurium 1,4,[5],12:i:— monophasicAnses Pork meat 168 2011LSAL06561 variant Typhimurium 1,4,[5],12:i:—monophasic Anses Veal meat 169 2012LSAL05317 variant Typhimurium1,4,[5],12:i:— monophasic Anses Turkey meat 170 2014LSAL02635 variantTyphimurium 1,4,[5],12:i:— monophasic Anses Gallus gallus 1712014LSAL03913 variant meat Typhimurium 1,4,[5],12:i:— monophasic AnsesPoultry Feed 172 2011LSAL04983 variant Typhimurium 1,4,[5],12:i:—monophasic Anses cattle feed 173 2012LSAL03407 variant Typhimurium1,4,[5],12:i:— monophasic Anses animal blood 174 2012LSAL03874 variantproducts Typhimurium 1,4,[5],12:i:— monophasic Anses cattle feed 1752015LSAL00792 variant Typhimurium 1,4,[5],12:i:— monophasic Anses beefmeat 176 2013LSAL02030 variant (carcass) Typhimurium 1,4,[5],12:i:—monophasic Anses pork 177 2015LSAL01461 variant (carcass) Typhimurium1,4,[5],12:i:— monophasic Anses turkey 178 2013LSAL03886 variant(carcass) Typhimurium 1,4,[5],12:i:— monophasic Anses chicken 1792016LSAL00194 variant (carcass)

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All patents, patent applications, internetsources, and other published reference materials cited in thisspecification are incorporated herein by reference in their entireties.Any discrepancy between any reference material cited herein or any priorart in general and an explicit teaching of this specification isintended to be resolved in favor of the teaching in this specification.This includes any discrepancy between an art-understood definition of aword or phrase and a definition explicitly provided in thisspecification of the same word or phrase.

1. A method of selectively detecting the presence of SalmonellaTyphimurium in a sample, the method comprising: (a) providing a reactionmixture comprising a suitable primer pair for amplification of asequence 95% homologous to SEQ ID NO:1 or a portion thereof; (b)performing PCR amplification of the nucleic acids of the sample usingthe reaction mixture of step (a); and (c) selectively detecting thepresence of Salmonella Typhimurium by detecting the amplified nucleicacids.
 2. The method of claim 1, wherein the reaction mixture comprisesa primer pair for amplification of a sequence 97% homologous to SEQ IDNO:1 or a portion thereof.
 3. The method of claim 1, wherein thereaction mixture comprises a primer pair for amplification of a sequence99% homologous to SEQ ID NO:1 or a portion thereof.
 4. The method ofclaim 1, wherein the reaction mixture comprises a primer pair foramplification of SEQ ID NO:1 or a portion thereof.
 5. The method ofclaim 1, wherein the reaction mixture comprises a primer pair foramplification of a sequence 95% homologous to SEQ ID NO:2 or a portionthereof.
 6. The method of claim 5, wherein the reaction mixturecomprises a primer pair for amplification of a sequence 97% homologousto SEQ ID NO:2 or a portion thereof.
 7. The method of claim 5, whereinthe reaction mixture comprises a primer pair for amplification of asequence 99% homologous to SEQ ID NO:2 or a portion thereof.
 8. Themethod of claim 5, wherein the reaction mixture comprises a primer pairfor amplification of SEQ ID NO:2 or a portion thereof.
 9. The method ofclaim 1, wherein the reaction mixture comprises a primer pair foramplification of a sequence 95% homologous to SEQ ID NO:3 or a portionthereof.
 10. The method of claim 9, wherein the reaction mixturecomprises a primer pair for amplification of a sequence 97% homologousto SEQ ID NO:3 or a portion thereof.
 11. The method of claim 9, whereinthe reaction mixture comprises a primer pair for amplification of asequence 99% homologous to SEQ ID NO:3 or a portion thereof.
 12. Themethod of claim 9, wherein the reaction mixture comprises a primer pairfor amplification of a sequence of SEQ ID NO:3 or a portion thereof. 13.The method of claims 10 to 12, wherein the primer pair for amplificationof the nucleic acid region of SEQ ID NO:3 comprises SEQ ID NO:4 and SEQID NO:5.
 14. The method of any of claims 1 to 13, wherein the reactionmixture further comprises a probe for the nucleic acid region to bedetected.
 15. The method of claim 14, wherein the probe comprises adetectable label.
 16. The method of claim 14 or 15, wherein the probecomprises SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.
 17. Themethod of any of claims 14 to 16, wherein the probe is selected from thegroup consisting of a double-stranded probe, a TAQMAN™ probe, amolecular beacon probe, a dual hybridization probe, an ECLIPSE™ probe,and a SCORPION™ probe.
 18. The method of claim 1, wherein the detectingthe presence of Salmonella Typhimurium comprises sequencing theamplified nucleic acids.
 19. The method of claim 1, wherein step (b) isperformed in partitions.
 20. A primer pair comprising the polynucleotidesequences as set forth in SEQ ID NO:4 and SEQ ID NO:5.
 21. A probecomprising the polynucleotide sequences set forth in SEQ ID NO:6 and SEQID NO:7.
 22. A probe comprising the polynucleotide sequences set forthin SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.
 23. Anisolated polynucleotide comprising a polynucleotide sequence having atleast 95% sequence identity based on the BLASTN method of alignment tothe polynucleotide sequence set forth in SEQ ID NO:1.
 24. The isolatedpolynucleotide of claim 23, wherein the isolated polynucleotide sequencecomprises a polynucleotide sequence set forth in SEQ ID NO:1.
 25. Anisolated polynucleotide comprising a polynucleotide sequence having atleast 95% sequence identity based on the BLASTN method of alignment tothe polynucleotide sequence set forth in SEQ ID NO:2.
 26. The isolatedpolynucleotide of claim 25, wherein the isolated polynucleotide sequencecomprises a polynucleotide sequence set forth in SEQ ID NO:2.
 27. Anisolated polynucleotide comprising a polynucleotide sequence having atleast 95% sequence identity based on the BLASTN method of alignment tothe polynucleotide sequence set forth in SEQ ID NO:3.
 28. The isolatedpolynucleotide of claim 27, wherein the isolated polynucleotide sequencecomprises a polynucleotide sequence set forth in SEQ ID NO:3.
 29. A kitfor the detection of Salmonella Typhimurium in a sample, the kitcomprising a primer pair comprising SEQ ID NO:4 and SEQ ID NO:5.
 30. Thekit of claim 29, further comprising a probe comprising SEQ ID NO:6 andSEQ ID NO:7.
 31. The kit of claim 29, further comprising a probecomprising SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. 32.The kit of claim 30 or 31, further comprising at least one componentselected from a lysis reagent, a DNA polymerase, dNTPs, a buffer, anegative control, a positive control, and instructions for performing amethod to detect the presence of Salmonella Typhimurium in a nucleicacid sample.